Summary of Work: This project is concerned with the structural and functional aspects of the DNA polymerase III epsilon subunit, the product of the dnaQ gene. Epsilon is a multifunctional protein that plays an essential part in the pol III holoenzyme complex. It is a component of the tightly-bound pol III core enzyme that, besides epsilon, is comprised of the alpha (= polymerase) subunit and theta subunit (of unknown function). Epsilon contains the 3' exonuclease function that serves as the polymerase proofreader. In addition, it tightly binds both the alpha and theta subunits and, on that basis, likely fulfills a structural function as well. The importance of the catalytic and structural functions is evidenced by the conditional lethality of dnaQ mutator mutants that are specifically defective in proofreading activity or dnaQ mutator mutants resulting from a dnaQ deletion. We have begun the structure-function analysis of epsilon by a detailed analysis of a series of dnaQ mutator mutants previously isolated in our laboratory. Specifically, we have (i) sequenced the mutant genes to reveal the location and nature of the responsible mutations, (ii) determined the magnitude and specificity of the mutator effects, (iii) determined whether these mutations are either recessive or dominant in a genetic complementation assay, and (iv) measured the interaction of the mutant epsilon subunits with the alpha and theta subunits using the yeast two-hybrid system. The combined data have provided new insights in the functional organization of e, including the significance of three conserved Exo motifs, of which one is quite distinct of the Exo motifs found in most other proofreading exonucleases. In addition, our evidence suggests that the C terminal part of e is a separate (sub)domain responsible for interaction to the polymerase subunit.